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Degradation of propanil by Acinetobacter baumannii DT immobilized in

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    Physical s
    ciences
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    Chemistry
    8
    SEPTEMBER 2022
    Volume 64 Number 3
    Introduction
    The proper use of herbicides in agricultural production
    saves money, time and labor, especially considering they
    have become indispensable in today’s agricultural sector.
    Propanil is a contact herbicide [1], which is used globally.
    This herbicide inhibits the photosynthesis of broadleaf weeds
    that leads to leaf chlorosis and the subsequent necrosis of
    leaves and other organs [2]. The herbicide is mainly applied
    to rice fields, which results in surface water, groundwater,
    and soil contamination [3-5]. Propanil has been detected
    at concentrations up to 3.6 mg/l in water [5], while the
    acceptable level of propanil in drinking water is 0.1 μg/l [6].
    Propanil is usually mixed with butachlor to increase the
    efficiency of weed eradication [7]. The negative effects of
    butachlor on soil microorganisms have also been studied
    [8-10]. Therefore, the biodegradation of propanil may be
    influenced by the presence of butachlor. However, few
    reports describing the effects of butachlor on the degradation
    of propanil have been published [11].
    Alginate is a natural polymer universally employed to
    immobilize cells in biodegradation experiments because
    alginate is relatively mild, chemically inert, inexpensive,
    and non-toxic [12]. Several bacteria and fungi such as
    Fusarium oxysporum
    [13],
    Paracoccus
    sp. FLN-7
    [14],
    Ochrobactrum
    sp. PP-2 [15], and
    Spirosoma sordidisoli
    TY50
    T
    [16] have been isolated. However, to our knowledge,
    no publication has yet described the degradation of propanil
    by microorganisms immobilized in alginate.
    In our previous report, propanil degradation by
    A. baumannii
    DT during the exponential growth phase with
    low cell density was determined [11, 17]. In this study,
    propanil degradation by
    A. baumannii
    DT immobilized in
    alginate and condensed counterparts is compared.
    Materials and methods
    Cultivation media
    The mineral medium (MM) used in this study was
    prepared according to Ha Danh Duc (2017) [18]. The MM
    contained (in grams per liter) Na
    2
    HPO
    4
    , 2.79; KH
    2
    PO
    4
    ,
    1.00; (NH
    4
    )
    2
    SO
    4
    , 1.00; MgSO
    4
    ·H
    2
    O, 0.20; and 1.00 ml of
    trace mineral solution. The trace mineral solution consisted
    of (in grams per liter) H
    3
    BO
    3
    , 0.30; CoCl
    2
    ·6H
    2
    O, 0.20;
    ZnSO
    4
    ·7H
    2
    O, 0.10; Na
    2
    MoO
    4
    ·2H
    2
    O, 0.03; MnCl
    2
    ·4H
    2
    O,
    0.03; NiCl
    2
    ·6H
    2
    O, 0.02; and CuCl
    2
    ·2H
    2
    O, 0.01. The pH of
    the MM was adjusted to 7.0±0.1. Ammonium sulfate (0.1%,
    w/v) and succinate (0.1%, w/v) were supplemented as a
    Degradation of propanil by
    Acinetobacter baumannii
    DT
    immobilized in alginate
    Danh Duc
    Ha
    *
    , Thi Oanh Nguyen
    Dong Thap University
    Received 5 October 2020; accepted 4 January 2021
    *
    Corresponding author: Email: [email protected]
    Abstract:
    The herbicide propanil has been widely applied in Vietnam and around the world to control weeds. In this
    study,
    Acinetobacter baumannii
    DT isolated from soil was used to determine propanil degradation. Degradation
    experiments were carried out with condensed cells at 10
    9
    CFU/ml with both free and immobilized forms of the
    bacteria. Propanil degradation rates by bacteria immobilized in alginate were higher than those of free cells
    at the same concentrations. The degradation curve as a function of concentration fit well to the degradation
    kinetics described by the Edwards model with a maximum degradation of 0.034±0.003 mM/h for free cells and
    0.053±0.005 mM/h for immobilized cells. Moreover, the immobilized bacteria could tolerate higher propanil
    concentrations and degrade propanil in a well-known herbicide more effectively compared to the freely
    suspended bacteria. These results demonstrate that
    A. baumannii
    DT immobilized in alginate is suitable for
    degradation of propanil in herbicides.
    Keywords:
    Acinetobacter baumannii
    DT, degradation, immobilized cells, kinetics, propanil.
    Classification number:
    2.2
    DOI : 10.31276/VJSTE.64(3).08-12
    Physical sciences
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    SEPTEMBER 2022
    Volume 64 Number 3
    nitrogen and carbon source, respectively. The medium was
    sterilized for 15 min at 121°C and stored at 4°C until further
    use. Pure propanil (99.6%) and herbicide with the trade
    name Cantanil 550EC (Thanhson Agrochem Company,
    Vietnam) were used. Cantanil 550EC contained 275 mg/l
    of propanil, 275 mg/l of butachlor, and adjuvant was used to
    determine the biodegradation of propanil.
    Resting cells and immobilization method
    To prepare for immobilization,
    A. baumannii
    DT was
    cultured in MM amended with ammonium sulfate and
    succinate medium for 12 h. Bacteria were collected by
    centrifugation at 10,000 rpm for 5 min. Cell pellets were
    washed twice with sterile MM. The mixture was then re-
    suspended in MM used for degradation by free cells (resting
    cells) while 2×MM was used for immobilization.
    The immobilization process was carried out according to
    a previous report [19] with modifications. The concentrated
    bacterial solution was mixed with a sterilized solution
    of Ca-alginate and glycerol to give final cell numbers
    of approximately 10
    9
    CFUs/ml, 3% alginate, and 10%
    glycerol. The solution was carefully blended and dripped
    into a solution containing 3% CaCl
    2
    (w/v) using a syringe.
    The beads formed in the solution were stirred for 1 h using a
    magnetic bar and then stored for 24 h at 4
    o
    C in this solution.
    The beads were collected and washed twice with MM before
    being used in experiments.
    Propanil degradation by resting cells and immobilized
    cells
    The degradation processes of propanil were carried out
    in MM with 10
    9
    CFUs/ml of both freely suspended and
    immobilized bacteria. Propanil was dissolved in absolute
    ethanol at 0.1 M and used as a stock solution. The degradation
    was carried out at propanil concentrations ranging from
    0.1 to 0.7 mM. The degradation rates at various propanil
    concentrations were determined and expressed in mM/h.
    Nonlinear regression analysis was used to fit the trends of
    the degradation process. The obtained data were best fit by
    kinetic models that incorporate the equation for logarithmic
    degradation such as the V.H. Edwards model (1970) [20]
    given by V=V
    max
    [e
    (-S/Ki)
    ‒e
    (-S/Ki)
    ] or the Haldane equation
    [21] V=(V
    max
    S)/(K
    s
    +S+S
    2
    /K
    i
    ) where V is the degradation
    velocity, V
    max
    is the maximum degradation rate, S is the
    propanil concentration, K
    i
    is the inhibition coefficient, and
    K
    s
    is the half-saturation coefficient. The kinetic parameters
    were then calculated based on linear regression fitting of
    the H. Lineweaver, D. Burk (1934) [22] plot or double
    reciprocal plot. The Dixon plot was used to determine the
    inhibition constant in which the reciprocal of the velocity
    (1/V) was plotted against the inhibitor concentrations [
    i
    ].
    For the degradation of propanil in herbicide, Cantanil
    550EC was added to 0.1 mM of propanil. The effects of
    butachlor on propanil degradation were also carried out.
    Pure butachlor or butachlor in the herbicide Cantanil 550EC
    were used at the same quantity (weight) as propanil. The
    incubation processes were conducted at room temperature
    (from 28 to 31
    o
    C) with a shaking speed of 150 rpm.
    Long-term storage condition
    For long-term storage, resting and entrapped bacteria
    were stored in Corning™ polyethylene terephthalate
    centrifuge tubes in the dark at 4
    o
    C. After 2 months of storage,
    both free and immobilized bacteria were kept at room
    temperature for 2 h before determining cell survival and
    biodegradation. The results were compared with bacteria
    numbers and degradation rates of fresh ones.
    The number of viable bacterial cells in an alginate bead
    has been described by M. Schoebitz, et al. (2012) [23] with
    some modification. The beads (1.0 g) were transferred to 10
    ml of sterile sodium citrate (6%, w/v) to dissolve at 30
    o
    C on
    a rotary shaker for 30 min. Then, the solution was serially
    diluted with MM and spread on an agar plate containing
    MM supplemented with ammonium sulfate and succinate.
    For the enumeration of non-immobilized cells, the bacteria
    solution was serially diluted and also spread on the plates.
    The number of survival bacteria was determined based on
    colonies emerging after being incubated for 24 h at 30
    o
    C in
    an incubator.
    Analytical method
    Propanil concentrations were measured using reverse
    phase high performance liquid chromatography (HPLC)
    (LC-10AD, Shimadzu, Japan) with a C18 column (5
    μm, 250×4.6 mm; Hyperclone, Phenomenex, USA) at
    an absorbance of 240 nm. A mixture of acetonitrile and
    ultrapure water (7:3, v/v) served as a mobile phase at a flow
    rate of 1 ml/min.
    Statistical analysis
    The obtained data are shown as the mean ± standard
    deviation (SD). Duncan’s multiple range test in the SPSS
    program (version 22.0) were used to determine differences
    among the treatments (p<0.05).
    Results and discussion
    Propanil degradation by A. baumannii DT
    A. baumannii
    DT isolated from soil can degrade propanil
    and 3,4-dichloroaniline [11].
    A. baumannii
    DT was shown
    to utilize propanil as a sole carbon and nitrogen source in
    a previous report [11]. However, supplementation with
    succinate and ammonium sulfate increased the degradation
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    rates [11]. So, all experiments in this work were carried out
    with the amendment of these co-substrates.
    The degradation by free bacteria and their immobilized
    counterparts were compared. The results showed that the
    degradation rates of propanil by immobilized cells were
    significantly higher than those of freely suspended cells at the
    same concentrations. At 0.5 mM, the complete degradation
    of propanil by immobilized bacteria was accomplished
    after 20 h while propanil degradation by free cells was not
    complete until after 24 h (Fig. 1). No propanil was lost in the
    control without alginate beads and bacteria, while the beads
    absorbed about 12% of the substrate after 24 h.
    and
    also
    sp read
    on
    the plates.
    The
    number
    of survival
    ba cteria
    wa
    s determined
    based
    on colonies
    emergi
    ng a
    fter being in
    cubated
    for
    24 h at 30
    o
    C in
    an incu
    bator.
    Analytical
    metho
    d
    Propanil
    conc
    entrations
    were
    measured
    using
    reverse
    phase
    high
    performance
    liquid
    chromat
    ography
    (HPLC)
    (LC
    -10
    AD,
    Shimadzu,
    Japa
    n)
    with
    a C18
    column
    (5
    μm,
    250
    ×4.6
    mm;
    Hy
    perclone,
    Phenome
    nex
    , USA)
    at
    an
    absor
    ban
    ce
    of
    240
    nm.
    A
    mixture
    of
    ace
    tonitrile
    and
    ultrapure
    water
    (7:3,
    v/v)
    serv
    ed
    as
    a mobile
    phas
    e at
    a
    flow rate of
    1 ml/m
    in.
    Statistical
    analys
    is
    The
    obtained
    data
    are
    shown
    as
    the
    mean
    ±s tandard
    deviat
    ion
    (SD)
    . Duncan’s
    multiple
    range
    test
    in the
    SPSS
    progra
    m
    (version
    22.0)
    were
    us ed
    to
    determin
    e
    differences a
    mong t
    he t
    reatments (
    p<0.05).
    Results a
    nd dis
    cussio
    n
    Propanil
    degrad
    ation
    by A. bau
    manni
    i DT
    A.
    baum
    annii
    DT
    isolated
    fr om
    soil
    can degrade
    prop
    anil
    and
    3, 4-
    dichloroani
    line
    [11].
    A.
    bauma
    nnii
    DT
    wa
    s sh own
    to utilize
    prop
    anil
    as
    a sole
    carbon
    and
    nitrogen
    source
    in
    a
    previous
    repor
    t [11].
    However,
    sup
    plementatio
    n
    with
    succinate
    and
    ammo
    nium
    sulfate
    increase
    d
    the
    degradat
    ion
    ra tes
    [11].
    So,
    al l
    experiment
    s in th
    is wor
    k were carrie
    d out w
    ith the amen
    dment of
    thes
    e co
    -substra
    tes.
    The
    degradat
    ion
    by
    free
    bacteria
    an d
    their
    immobilize
    d
    counte
    rparts
    were
    compared.
    The
    results
    showed
    that
    the
    degra
    dation
    rates
    of
    propani
    l by
    immobi
    lize
    d
    cells
    were
    signific
    antl
    y
    higher
    than
    those
    of
    freely
    suspende
    d
    cells
    at
    the
    same
    concentrati
    ons.
    At
    0.5
    mM,
    the
    com
    plete
    degradation
    of
    pro
    panil
    by
    im
    mobilized
    Fig.
    1.
    Prop
    anil
    deg
    radation
    by
    free
    an
    d
    im
    mobilized
    cells
    (solid
    line
    s).
    Th
    e
    controls
    (da
    shed li
    nes)
    were also ru
    n in para
    lle
    l.
    0
    0.1
    0.2
    0.3
    0.4
    0.5
    0
    5
    10
    15
    20
    25
    Pr
    opanil
    (m
    M)
    Tim
    e (hou
    r)
    Fre e cell
    s
    Imm
    obil
    iz ed c
    ell s
    Fig. 1.
    Propanil degradation by free and immobilized cells (solid
    lines).
    The controls (dashed lines) were also run in parallel.
    Degradation kinetics of freely suspended and
    immobilized cells.
    The degradation rates at various concentrations showed
    that propanil degradation by freely suspended resting cells
    and immobilized cells in alginate beads both followed the
    Edwards model, in which degradation rates increase at
    low concentrations but are inhibited at high concentrations
    (Fig. 2). The degradation rates caused by the resting cells
    and their immobilized counterparts increased by 0.3 and 0.4
    mM, respectively.
    Fig. 2. Relation between propanil concentrations and degradation
    rates of free resting and immobilized cells.
    The parameters of d
    egradation kinetics were extracted
    from the Edwards equation and are shown in Table 1.
    The maximum degradation
    rate of immobilized bacteria
    was statistically higher than that of the free counterparts.
    The inhibition coefficient of degradation, which is the
    concentration required to produce half maximum inhibition,
    by free cells was also significantly lower compared to the
    immobilized cells. However, the half-saturation coefficients,
    which are the reaction rates at half-maximum, of free cells
    and immobilized bacteria were not statistically different.
    These results showed that immobilized bacteria were more
    tolerant to the toxicity of propanil. Similarly, the degradation
    of toluene and chlorotoluene by
    Comamonas testosterone
    KT5 immobilized in alginate was higher than that of freely
    suspended cells [24]. Propanil is a toxic compound, so the
    substrate inhibition occurred. The enhanced degradation
    of immobilized cells was because bacteria were protected
    by alginate matrix. In alginate beads, substrate has to
    diffuse through the immobilization barrier, and will then be
    available for the cells to utilize, which reduced the toxicity
    to bacteria.
    Table 1. Degradation parameters of free suspended cells and
    immobilized cells.
    Parameters
    Free suspended cells
    Immobilized cells
    Maximum degradation rate (mM/hour)
    0.034±0.003
    a
    0.053±0.005
    b
    Half-saturation coefficient (mM)
    0.249±0.022
    a
    0.296±0.030
    a
    Inhibition coefficient (mM)
    3.50±0.24
    a
    4.32±0.41
    b
    Data are shown as mean ±SD and different superscript letters (a and
    b) denote a significant difference (p<0.05) between treatments in a
    line based on Duncan’s test, whereas the same letter indicates no
    significant difference.
    In our previous report, V
    max
    , K
    s
    and K
    i
    of propanil
    utilization by freely suspended bacteria in the exponential
    growth phase were 0.027±0.003 mM/h, 0.16±0.02 mM and
    0.33±0.03 mM, respectively [11]. In this study, the V
    max
    and
    K
    s
    of concentrated bacteria were comparable higher than
    those of low bacteria density as described in the previous
    report [11]. The degradation and growth rates of bacteria
    in the exponential growth phase showed the activities of
    A. baumannii
    DT to propanil, while the degradation by
    concentrated bacteria provided a potential application to
    remediate the herbicide. Propanil detected in contaminated
    sites by E.G. Primel, et al. (2007) [5], of course, was
    mostly lower concentrations in this study.
    A. baumannii
    DT was investigated for degradation of various propanil
    concentrations, which showed the application ability to
    remediate in contaminated sites.
    Effects of butachlor on degradation of pure propanil
    and degradation of propanil in herbicide Cantanil 550EC
    Figure 3 shows that the percentage of propanil degradation
    for immobilized bacteria were significantly higher compared
    to those for free cells under the same conditions. The
    addition of pure butachlor and Cantanil 550EC decreased
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    the degr
    adation rates. Similarly, the effects of butachlor on
    propanil degradation and growth of
    A. baumannii
    DT have
    been described in previous studies [11, 17]. A number of
    trace herbicides contain both butachlor and propanil, but
    both substrates inhibited each other in biodegradation [17].
    The degradation was the lowest with the amendment of the
    herbicide Cantanil 550EC. These results can be explained
    by the negative effects from the adjuvants in the herbicide
    on the degradation propanil, which was also described by
    H.D. Duc, et al. (2020) [17].
    conditions. The addition of pure butachlor and Cantanil 550EC decreased the
    degradation rates. Sim
    ilarly, the effects of butachlor on propanil degradation and
    growth of
    A. baumannii
    DT
    have been
    described in previous studies [11, 17]. A
    number of trace herbicides contain both butachlor and propanil, but both substrates
    inhibited each other in biodegrad
    ation [17]. The degradation was
    the
    lowest with the
    amendment of
    the
    herbicide Cantanil 550EC. The
    se
    results
    can
    be explained
    by the
    negative effects from the
    adjuvants in the herbicide
    on
    the degradation propanil,
    which was also described by Duc, et al.
    (
    2020)
    [17].
    Fig. 3. Effects of butachlor on degradation of pure propanil and degradation of
    propanil in herbicide Cantanil 550EC.
    The degradation processes were carried out
    for 12 h at 0.1 mM propanil.
    Different letters (a, b
    , c, d
    and
    e
    )
    above the
    columns
    denote a significant difference (
    p
    <0.05)
    among treatments.
    Bacteria
    survival and propanil degradation for long
    term storage
    The
    number of live bacteria
    decreased
    after two month
    s of
    storage. The
    survival percentages of bacteria
    amended
    with glycerol were higher than those of
    treatments without the cryoprotectant (Fig. 4). These results indicated that using
    glycerol as a cryoprotectant agent reduced the adverse effects of
    the
    bacteria.
    The
    survival of free and entrapped bacteria was not statistically different
    under
    the same
    conditions.
    0
    20
    40
    60
    80
    100
    120
    Free cells,
    without
    butachlor
    Immobilized
    cells, without
    butachlor
    Free cells, with
    butachlor
    Immobilized
    cells, with
    butachlor
    Free cells,
    herbicide
    Immobilized
    cells, herbicide
    Propanil degradation (%)
    a
    b
    b
    c
    d
    e
    Fig. 3. Effects of butachlor on degradation of pure propanil
    and degradation of propanil in herbicide Cantanil 550EC.
    The
    degradation processes were carried out for 12 h at 0.1 mM propanil.
    Different letters (a, b, c, d and e) above the columns denote a significant
    difference (p<0.05) among treatments.
    Bacteria survival and propanil degradation for long-
    term storage
    The number of live bacteria decreased after two months
    of storage. The survival percentages of bacteria amended
    with glycerol were higher than those of treatments without
    the cryoprotectant (Fig. 4). These results indicated that using
    glycerol as a cryoprotectant agent reduced the adverse effects
    of the bacteria. The survival of free and entrapped bacteria
    was not stati
    stically different under the same conditions.
    Fig. 4.
    Survival of
    A. baumannii
    DT after two
    months’
    storage at 4
    o
    C.
    Different
    letters (a, b
    ,
    and c)
    above the columns denote a
    significant difference (
    p
    <0.05)
    among
    treatments.
    Table 2 shows that the degradation by immobilized bacteria was significantly
    higher than that
    of the
    resting cells, which was similar to the experiments described
    above. The addition of glycerol reduced pr
    opanil degradation from about 8.2 to
    15.6
    %
    at the beginning. However, the presence of glycerol resulted in
    a non
    statistical
    ly
    significant
    reduction of degradation after two months
    of
    storage. The degradation rates
    of the treatments with glycerol decreased
    from only 6.1 to 12%, while data of
    treatments without glycerol were reduced from 27.9 to 36.4%.
    The results show that the addition of glycerol reduced the adverse effects of
    bacteria.
    Previous reports also presented that the survival of entrapped
    microo
    rganisms
    is
    enhanced
    by
    the addition of glycerol
    [25, 26]
    . Glycerol is used as a
    cryoprotectant
    that
    can
    prevent
    ice
    crystal formation after penetration into the cells
    [27]. Another report shows that t
    he addition of glycerol
    protects the
    microorganisms,
    increases pore size in beads, and
    controls the structure of dried macrocapsules [26].
    Table 2. Propanil degradation by free cells and immobilized cells.
    The degradation
    processes were carried out for 10 h at 0.1 mM propanil.
    Propanil de
    gradation (%)
    Free suspended cells
    Immobilized cells
    At the
    beginning
    After two
    months
    At the
    beginning
    After two
    months
    Pure
    propanil
    Without glycerol
    92.1±4.2
    Dc
    55.5±8.1
    Ba
    98.4±1.5
    Cc
    68.4±7.9
    Bb
    With glycerol
    80.6±6.3
    Cab
    72.8±7.0
    Ca
    90.2±4.2
    Cb
    78.2±7.3
    Bab
    Herbicide
    Without glycerol
    43.4±5.5
    Bc
    15.5±4.3
    Aa
    63.4±6.1
    Bd
    30.4±5.2
    Ab
    With glycerol
    30.2±5.6
    Aa
    22.6±5.6
    Aa
    47.8±6.5
    Ab
    41.7±6.2
    Ab
    0
    20
    40
    60
    80
    100
    Free cells
    Free cells with
    glycerol
    Immobilized cells
    Immobilized cells
    with glycerol
    Bacteria survival (%)
    a
    a
    bc
    c
    Fig. 4.
    Survival of
    A. baumannii
    DT after two months’ storage
    at 4
    o
    C.
    Different letters (a, b, and c) above the columns denote a
    significant difference (p<0.05) among treatments.
    Table 2 shows that the degradation by immobilized
    bacteria was significantly higher than that of the resting
    cells, which was similar to the experiments described above.
    The addition of glycerol reduced propanil degradation from
    about 8.2 to 15.6% at the beginning. However, the presence
    of glycerol resulted in a non-statistically significant reduction
    of degradation after two months of storage. The degradation
    rates of the treatments with glycerol decreased from only
    6.1 to 12%, while data of treatments without glycerol were
    reduced from 27.9 to 36.4%.
    Table 2. Propanil degradation by free cells and immobilized cells.
    The degradation processes were carried out for 10 h at 0.1 mM
    propanil.
    Propanil degradation (%)
    Free suspended cells
    Immobilized cells
    At the
    beginning
    After two
    months
    At the
    beginning
    After two
    months
    Pure
    propanil
    Without glycerol
    92.1±4.2
    Dc
    55.5±8.1
    Ba
    98.4±1.5
    Cc
    68.4±7.9
    Bb
    With glycerol
    80.6±6.3
    Cab
    72.8±7.0
    Ca
    90.2±4.2
    Cb
    78.2±7.3
    Bab
    Herbicide
    Without glycerol
    43.4±5.5
    Bc
    15.5±4.3
    Aa
    63.4±6.1
    Bd
    30.4±5.2
    Ab
    With glycerol
    30.2±5.6
    Aa
    22.6±5.6
    Aa
    47.8±6.5
    Ab
    41.7±6.2
    Ab
    The lowercase superscript letters show statistically significant
    differences in the same row, while capitalized superscript letters indicate
    statistically significant differences among treatments within a column
    (p<0.05). Data are means of the results from at least three individual
    experiments, and mean values and standard deviations are shown.
    The results show that the addition of glycerol reduced the
    adverse effects of bacteria. Previous reports also presented
    that the survival of entrapped microorganisms is enhanced
    by the addition of glycerol [25, 26]. Glycerol is used as a
    cryoprotectant that can prevent ice crystal formation after
    penetration into the cells [27]. Another report shows that the
    addition of glycerol protects the microorganisms, increases
    pore size in beads, and controls the structure of dried
    macrocapsules [26].
    Conclusions
    A. baumannii
    DT immobilized in alginate showed
    more effective propanil degradation than free cells. It was
    confirmed that the chemical degradation over a wide range
    of concentrations by condensed free cells and immobilized
    cells followed the Edwards model with higher degradation
    rates and inhibition coefficients for immobilized bacteria.
    Besides, immobilized bacteria degraded propanil in an
    herbicide with higher rates than its free counterparts.
    Moreover, bacteria entrapped in alginate beads amended with
    glycerol reduced the adverse effects of long-term storage.
    These results indicate that
    A. baumannii
    DT immobilized in
    alginate can be applied to remediate propanil.
    COMPETING INTERESTS
    The authors declare that there is no conflict of interest
    regarding the publication of this article.
    conditions. The addition of pure butachlor and Cantanil 550EC decreased the
    degradation rates. Sim
    ilarly, the effects of butachlor on propanil degradation and
    growth of
    A. baumannii
    DT
    have been
    described in previous studies [11, 17]. A
    number of trace herbicides contain both butachlor and propanil, but both substrates
    inhibited each other in biodegrad
    ation [17]. The degradation was
    the
    lowest with the
    amendment of
    the
    herbicide Cantanil 550EC. The
    se
    results
    can
    be explained
    by the
    negative effects from the
    adjuvants in the herbicide
    on
    the degradation propanil,
    which was also described by Duc, et al.
    (
    2020)
    [17].
    Fig. 3. Effects of butachlor on degradation of pure propanil and degradation of
    propanil in herbicide Cantanil 550EC.
    The degradation processes were carried out
    for 12 h at 0.1 mM propanil.
    Different letters (a, b
    , c, d
    and
    e
    )
    above the
    columns
    denote a significant difference (
    p
    <0.05)
    among treatments.
    Bacteria
    survival and propanil degradation for long
    term storage
    The
    number of live bacteria
    decreased
    after two month
    s of
    storage. The
    survival percentages of bacteria
    amended
    with glycerol were higher than those of
    treatments without the cryoprotectant (Fig. 4). These results indicated that using
    glycerol as a cryoprotectant agent reduced the adverse effects of
    the
    bacteria.
    The
    survival of free and entrapped bacteria was not statistically different
    under
    the same
    conditions.
    0
    20
    40
    60
    80
    100
    120
    Free cells,
    without
    butachlor
    Immobilized
    cells, without
    butachlor
    Free cells, with
    butachlor
    Immobilized
    cells, with
    butachlor
    Free cells,
    herbicide
    Immobilized
    cells, herbicide
    Propanil degradation (%)
    a
    b
    b
    c
    d
    e

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